Immunophenotyping (Haematology Cell Markers)

Immunophenotyping (Haematology Cell Markers)

The laboratory is equipped with 2 Beckman Coulter Navios flow cytometers. Fluorochrome conjugated monoclonal antibodies are used to label cells. Antibody/Antigen reactions are recognisable via fluorescence emitted during laser excitation within the flow cytometer.

The haematology department currently provides a service for diagnosis and monitoring of PNH, and for quantitation of foetal maternal haemorrhage. These are described in further detail below.

All diagnostic flow cytometric tests for haematological malignancies such as leukaemia and lymphoma are now sent to the Clinical Immunology Department at Birmingham Medical School and are reported via the MIHRO service (Midland Integrated Reporting for Haemato-Oncology). This centre also handles requests for cytogenetics, molecular genetics and bone marrow morphology.

MIHRO requests

Please use the MIHRO request form which provides information on sample types required.

https://www.birmingham.ac.uk/facilities/clinical-immunology-services/services/handbook/index.aspx

MIHRO require samples to be labelled with 4 identifiers (Surname, firstname, hospital number/DOB, NHS number) to provide positive ID against the request form. Failure to adequately label may result in sample rejection.

Additionally, MIHRO requires that patient address, clinical details and consultant name must be provided.

Please avoid sending samples after 11:00am on Fridays as MIHRO do not offer a weekend service and samples need to be couriered.

Requests from non-haematology ward/clinic should be discussed with clinical haematology.

For advice contact

Laboratory Haematology, Cell Markers/Molecular haematology    0121 424 0704.

MIHRO   0121 414 4069

PNH and FMH requests (Non-MIHRO)

These are performed at BHH

Laboratory opening:

The laboratory is open Monday - Friday, 08:30 - 17:00. Latest time of sample receipt for guaranteed same day analysis is 14.00. Samples arriving later than this may be refrigerated until the next working day. Analysis may be performed following weekend storage providing cell viability is within acceptable limits and IQC checks are acceptable.

Samples should be transported to the laboratory as soon as possible after collection following the transport procedure.  For external requests, samples should be transported in suitable containers via courier and labelled for the attention of Cell Markers, Haematology BHH. Samples should not be sent in the post.

Contact:

Cell Markers:              0121 424 0704

Clinical advice:            please contact the on-call Consultant Haematologist via switchboard 0121 424 2000.

Requests will be accepted on any suitable request form provided the usual labelling criteria are met:

There must be a minimum of 3 identifiers which link the sample and the request form and should include the patient’s first name, patient’s surname and either hospital number, date of birth or NHS number.

PNH SCREEN

Paroxysmal Nocturnal Haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder resulting in a partial or total deficiency of proteins normally linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor.

Classical features are:

intravascular haemolysis

bone marrow failure

thrombotic tendency

There may also be abdominal pain, dysphagia, erectile dysfunction, renal failure, lethargy. It can often go undiagnosed for months or even years. Flow cytometry can provide a definitive diagnosis.

PNH screening can only be performed on peripheral blood (taken into EDTA). Marrow is unsuitable due to the presence of immature cells.

Investigation for PNH involves the analysis of antigens normally linked via the GPI anchor on red cells, neutrophils and monocytes. These include CD59 (red cells), CD24 and CD14 (neutrophils and monocytes respectively). Additionally, neutrophil and monocyte screening includes FLAER (FLuorescent AERolysin) which binds directly to GPI. Red cell analysis provides classification into PNH types I and II but clone size may not be representative due to transfusion and/or haemolysis. Neutrophil and/or monocyte analysis provides an accurate measure of clone size and is used to monitor the disorder over time.

Please indicate recent transfusion history on the request form.

PNH requests require a separate EDTA. If the patient is severely neutropenic please take two samples. Any recent transfusions should be highlighted on the request form.

See Test Database for information on sensitivity and sample requirements

FETO-MATERNAL HAEMORRHAGE

FMH by flow cytometry identifies and enumerates the bleed of foetal cells into maternal circulation following birth or other traumatic event. Please note that this screen is based on the principle that maternal samples are Rh D negative and baby/cord samples are Rh D positive. In ante-natal patients or patients who have had an IUD it may not be possible to identify a bleed if mother and baby have the same Rh D type. Also as the principle is based on staining using a fluorescent anti-D, patients who have received recent prophylactic anti-D may give reduced values due to antigen blocking by prophylactic anti-D.

To ensure the correct dose of anti-D is administered we report a suggested dosage taking into account the uncertainty of measurement of FMH by flow in our laboratory. The reported bleed (in mls) is the value obtained by flow cytometry. The suggested dosage of anti-D is based on the reported bleed plus the uncertainty of measurement. 

Current guidelines state that FMH quantitation by flow cytometry should be performed on any bleed greater than 2mls as identified by the Kleihauer stain (performed in the Blood Bank).  

FMH requests should be made following current BCSH guidelines. Please phone the laboratory prior to sending and ensure contact details are provided plus any relevant history. An FMH request form may be downloaded here if required.

For any further advice and to arrange a test please telephone the laboratory on 0121 424 0704